enzyme immunoassay the eia is a type of nonisotopic immunoassay in which enzymes, coenzymes,...
TRANSCRIPT
![Page 1: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/1.jpg)
![Page 2: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/2.jpg)
![Page 3: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/3.jpg)
Enzyme ImmunoassayThe EIA is a type of nonisotopic immunoassay in
which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used as labels
The major prerequisite is that the antigen or antibody must be linked to an enzyme without destroying the immunologic or enzymatic activity of the antigen-antibody complex
![Page 4: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/4.jpg)
• valuable tools for use in clinical labs• can measure biological materials (antibodies or antigens)• inexpensive, rapid, quantitative, specific• sensitive (pg/ml)• expensive equipment not required (but helps)• can be automated
![Page 5: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/5.jpg)
Major labeled immunoassays
RIAEIAChemlumIFA
![Page 6: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/6.jpg)
Key terms
AnalyteImmunoassayLableLigandReactantseperation stepSolid phasesubstrate
![Page 7: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/7.jpg)
BASIC FORMAT
Solid phase = 96 / 384-well microplate
![Page 8: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/8.jpg)
Enzyme labelsHorseradish peroxidase (Horseradish )alkaline phosphatase (calf intestine)Glucose oxidase (Aspergillus niger)B-galactosidase (Ecoli):
- Are not naturally in the patient's sample
- High specific activity- Stability
![Page 9: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/9.jpg)
Enzymes used in immunoassay systemsmust be stableavailable in a highly purified statehave a high turnover rateundergo minimal interference by substances
likely to be in the test solutionand be specific for the substrate
![Page 10: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/10.jpg)
Types of protocols in labeled immunoassay
Competitive bindingNon-competitive bindingSandwich techniqueOne-step assayHomogeneous reactionHeterogeneous reaction
![Page 11: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/11.jpg)
Analyte = antibody Analyte = antigen
Incubate, wash
1. Coat solid phase withantigen when analysing antibody
antibody when analysing antigen
![Page 12: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/12.jpg)
2. Block free binding sites. Incubate. Wash.
Analyte = antibody Analyte = antigen
![Page 13: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/13.jpg)
3. Add sample. Incubate. Wash
Analyte = antibody Analyte = antigen
![Page 14: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/14.jpg)
Enzyme labelled (rabbit) anti-(human) Ig
(Second antibody)anti-(rabbit) Ig-enzyme
EE
Horse radish peroxidase (HRP)Alkaline phosphatase
![Page 15: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/15.jpg)
See Sigma catalogue for list of conjugates and substrates
Orthophenylene diamine Tetramethyl hydrochloride (OPD) benzidine (TMP)
Horse radish peroxidase (HRP) Orange, 490 nm Yellow, 450 nm Spectrophotometer
![Page 16: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/16.jpg)
The most widely used enzyme in EIA is horseradish peroxidase (HRP).
The substrate of HRP is hydrogen peroxide (H2O2) and the product is oxygen
This oxygen produced during the reaction is used to oxidize a reduced, colorless chromagen (usually reduced orthophenylenediamine)
The final product, oxidized orthophenylenediame, has a brown color
![Page 17: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/17.jpg)
Paranitrophenyl phosphate (PNP) Methyl umbelliferol phosphate
Alkaline phosphatase
Yellow, 405 nm Methyl umbelliferone Spectrophotometer
365 nm 445 nm Fluorimeter
![Page 18: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/18.jpg)
5. Add substrate
6. Incubate, stop, measure colour change
Colourless
ENZYME
OD
CONCENTRATION
![Page 19: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/19.jpg)
E
Directly conjugated developingantibody may give weak signal
![Page 20: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/20.jpg)
unlabelled (rabbit) anti-(human) Ig followed by
anti-(rabbit) Ig-enzyme
EE
a anti-rabbit labeled antibody
![Page 21: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/21.jpg)
Biotin-labelled anti-Ig followed by
streptavidin-enzyme
E-S B S-E
ES
streptavidin-enzymesteptavidin-nzyme
streptavidin-enzyme
streptavidin-enzyme
Streptavidin-Enzyme
![Page 22: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/22.jpg)
INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES
E E E
3. Anti-(human) Ig-enzyme
2. Sample (human) antibody
1.Antigen
Toxoplasma IgG
![Page 23: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/23.jpg)
INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES
E E E
4. Substrate
3. Anti-(human) Ig-enzyme
2. Sample (human) antibody
1. Antigen
![Page 24: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/24.jpg)
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
2. Impure antigen eg tissue homogenate
1. Specific antibody
![Page 25: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/25.jpg)
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
3. Wash pure antigen
2. Impure antigen
1. Specific antibody
![Page 26: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/26.jpg)
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
E E5. Anti-human Ig-enzyme
4. Sample (human antibody)
3. Wash pure antigen
2. Impure antigen
1. Specific antibody
![Page 27: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/27.jpg)
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
6. Substrate5. Anti-human Ig-enzyme
4. Sample (human antibody)
3. Wash pure antigen
2. Impure antigen
1. Specific antibody
![Page 28: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/28.jpg)
ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
eg. hormones drugs tumour antigens cytokines
1. Anti-analyte
![Page 29: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/29.jpg)
ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
1. Anti-analyte
2. Sample
3. anti-analyte- biotin followed by streptavidin-enzyme
E-S B S-E
ES
E S E-S B S-E
![Page 30: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/30.jpg)
ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
1. Anti-analyte
2. Sample
4. Substrate
3. anti-analyte-biotin followed by streptavidin-enzyme
![Page 31: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/31.jpg)
1
2
2
HEALTH
CANCER VIRUSES
MYCOBACTERIAHELMINTHS
ASTHMA, ALLERGYLUPUS
RHEUMATOID ARTHRITISMULTIPLE SCLEROSIS UVEITISDIABETES
IL2IL12IFNTNF
IL4 IL5 IL6 IL10
CMI(AB)
Type 1 Type 2
AB(CMI)
CYTOKINES
![Page 32: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/32.jpg)
MICROARRAYEg. Novagen ProteoPlex
Std 1
Std 3 Std 4
Std 2
Std 6
#2
#4
#6
#8
#10
Std 5
#1
#3
#5
#7
#9
IL1
IL2
IL5
IL7
IL10
IL16
gmCSF
TNF
IL1
IL4
IL6
IL8
IL12
IL18
IFN
TGF
Quadruplicate capture anti-cytokine antibody spotsOverlay with standards, samples. Incubate, washAdd fluorophore-detection antibody. Incubate, washScan
![Page 33: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/33.jpg)
Erroneous Results Caused by antibodies in immunoassay
The application of Abs as an analytical reagent
MoAb novel detection signals
In spite of technologic evolution during past 30 years, still :
False False
Positive Negative
![Page 34: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/34.jpg)
![Page 35: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/35.jpg)
![Page 36: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/36.jpg)
Heterophil antibodiesEndogenous antibodies produced against poorly defined antigens
Can react with several animal species (mouse is most common)
Usually react with Fc
IgG or IgM but can be IgA, IgE
Frequency : up to 40% (0.05% may present clinical
significance)
Produced by enviromental contact, transplacental passage or viral infection
![Page 37: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/37.jpg)
Human anti-animal antibodies (HAAA)Response to parenteral administration of animal MoAb
Following radioimaging, cancer therapy, transplant immunotherapy
High concentration, high avidity, epitope specific
![Page 38: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/38.jpg)
The steps to minimize interferences 1. The use of chimeric Abs (animal Fab + human Fc)
2. Using other methods based upon different MoAb
3. Removal of unusual Ab by PEG
4. Making dilutions usually produce non linear
results
5. Commercial mixture of animal proteins for pre-analysis incubation
![Page 39: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/39.jpg)
Antibody cross reactivity Although MoAb enhances specificity , still remains a source of error
Drug assay vs active, non active metabolites
Cross-reactants 1- positive interferent bias 2- Unexpected negative interferent
![Page 40: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/40.jpg)
Hook effectUnexpected fall in the amount of analyte resulting in the gross under-estimation of the analyte
Particularly in sandwich immunoassays when sample contain extremely high level
Upon further dilution, the result will be out of range
Most commonly occurred in measurement of IgE, hCG, Ferittin, tumor marker, infectious Ag-Ab
![Page 41: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/41.jpg)
Choices of antibodiesPolyclonal
IgG fraction or F (ab') 2 fragments
Affinity purified polyclonal
Monoclonal
![Page 42: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/42.jpg)
![Page 43: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/43.jpg)
How to deal with Hook effect1. Run all samples in duplicate dilution
2. Ensure for adequate washings
3. Know the level of Hook phenomenon according to manufacturer suggestion
4. Good communication with clinicians
![Page 44: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/44.jpg)
Rheumatoid Factor, RFAn IgM that act to IgG Fc70% RA patientsMay bind to rabbit, sheep, goat and mouse IgSpecial technical problem for IgM quantitationFalse positivity is common, but competitive inhibitor may cause false negative
![Page 45: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/45.jpg)
How to evade RF interference1. Using isolated IgM preparation
2. Addition of precipitating anti-IgG
3. Removing the RF using an aggregated IgG4. Using IgM capture assay problem can still occur: all IgM-specific antibody should be
evaluated cautiously.
![Page 46: Enzyme Immunoassay The EIA is a type of nonisotopic immunoassay in which enzymes, coenzymes, fluorigenic substrates, or enzyme inhibitors are used](https://reader033.vdocuments.us/reader033/viewer/2022061305/55145b36550346494e8b5623/html5/thumbnails/46.jpg)